LOCUS       sequence-257714-        2470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered sgRNA-LEB5 generator including Anderson promoter J23100 
            for golden gate assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 346 to 380)
  AUTHORS   Liu Y, Wan X, Wang B
  TITLE     Engineered CRISPRa enables programmable eukaryote-like gene 
            activation in bacteria.
  JOURNAL   Nat Commun. 2019 Aug 26;10(1):3693. doi: 10.1038/s41467-019-11479-0.
  PUBMED    31451697
REFERENCE   2  (bases 1 to 2470)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-08-26"; volume: "10(1):3693. doi"; pages: " 
            10.1038/s41467-019-11479-0"
COMMENT     Sequence Label: pLY134
FEATURES             Location/Qualifiers
     source          1..2470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..89
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(113..170)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     misc_feature    171..191
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     protein_bind    235..253
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    290..308
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     promoter        complement(346..380)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     misc_feature    458..479
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      482..525
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(529..547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(741..760)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             752..1567
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             752..1567
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(825..844)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1800..2388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2289..2308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
       61 cgaccgagcg cagcgagtca gtgagcgagg aagcctgcat aacgcgaagt aatcttttcg
      121 gttttaaaga aaaagggcag ggtggtgaca ccttgccctt ttttgccgga ctgcagcggc
      181 cgctactagt agaagacaac tagcttcaaa aaaagcaccg actcggtgcc acttgggccc
      241 ttcttcaggg cccaagttga taacggacta gccttatttg aacttgctag ggcccttctt
      301 cagggcccta gctctcaaac cacttatata ttcatccctc tagtagctag cactgtacct
      361 aggactgagc tagccgtcaa ctctagtacc tttggtcgaa aaaaaaagcc cgcactgtca
      421 ggtgcgggct tttttctgtg tttcctgtct tgtcttcctc tagaagcggc cgcgaattcc
      481 agaaatcatc cttagcgaaa gctaaggatt ttttttatct gaaattctgc ctcgtgatac
      541 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca agtggcactc
      601 gagctgatcc ttcaactcag caaaagttcg atttattcaa caaagccacg ttgtgtctca
      661 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
      721 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
      781 ctccaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
      841 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
      901 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
      961 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     1021 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     1081 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     1141 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     1201 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     1261 taatggctgg cctgttgaac aagtctggaa agaaatgcat aagcttttgc cattctcacc
     1321 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     1381 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     1441 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     1501 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     1561 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     1621 ggactcgagc tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1681 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1741 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1801 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1861 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1921 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     1981 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2041 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2101 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2161 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2221 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2281 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2341 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tgtggaaaaa cgccagcaac
     2401 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2461 ttatcccctg
//
